This invention relates to a method of stabilizing the enzyme arylacylamidase as well as a simplified enzymatic method and composition for the quantitation of anilide compounds, i.e. N-acylated primary aromatic amines or N-substituted acetamides including N-arylacetamides, such as acetaminophen (4-hydroxyacetanilide) in samples containing these drugs including biological fluids such as urine, plasma, serum or blood.
Acetaminophen, for example, is commonly used as an analgesic and antipyretic. It is found in many formulations promoted for the relief of pain, cough and colds. Because it can produce adverse side effects, its quantitation or estimation in cases of overdose is particularly important. Cases of overdose may lead to hepatic necrosis with possible fatal hepatic failure as reported in Ann. of Int. Med., 87, 202 (1977). The plasma concentration of acetaminophen is indicative of clinical evidence of liver damage. In cases of overdose known antidotes are administered. Therefore, the simplest and quickest method of testing for this material provides the greatest advantage to the patient.
Several chemical methods for the estimation of an anilide are known. These methods involve the addition of chemical reagents to the solution containing the anilide and the spectrophotometric determination of the resulting colored compound. Examples of these methods are described by J. H. Routh, et al., Clin. Chem. 14, 882 (1968), S. L. Tompsett, Ann. Clin. Biochem. 6, 81 (1969), J. P. Glynn, et al. Lancet 1, 1147 (1975) and G. S. Wilkinson, Ann.Clin. Biochem. 13, 435 (1976). In some of these methods, after the anilide is chemically hydrolysed by acids under a variety of conditions of temperature and time, the resulting aniline or aniline derivative formed is reacted with a substituted phenol or phenolic ether, such as ortho-cresol to give color which can be spectrophotometrically measured at 615 nm.
It has also been known for many years that several organisms produce enzymes (arylacylamide amidohydrolase or arylacylamidase) defined in group E. C. 3.5.1.13, capable of hydrolysing N-arylacylamides. Examples are R. P. Lanzilotta Ph.D. Thesis, Rutgers University, New Brunswick, N.J. 1968, N. E. Sharabi et al. App. Microb. 18, 369 (1969), D. J. W. Grant et al., Microbio. 8, 15 (1973). J. Alt et al. in J. of Gen. Microb. 87, 260 (1975) also found another bacterial strain of Pseudomonas (gram negative rods) namely Pseudomonas acidovorans ATCC 15668 which contains an arylamidase E. C. 3.5.1.13 which also hydrolyses anilides.
Moreover, it has been previously disclosed that the enzymatic hydrolysis of the anilide p-nitroacetanilide to an aniline can be measured spectrophotometrically at 405 nm. The spectrophotometric estimation of the anilide produced by another arylacylamidase enzyme was also reported in U.K. patent GB 2089978 B (1984), U.S. Pat. No. 414,327 (1983) and P. M. Hammond, et al. in Anal. Biochem. 143, 152 (1984). In these publications, hydrolysis of anilides is accomplished by an enzyme. The enzyme E. C. 3.5.1.13 described was derived from a Pseudomonas species, namely Pseudomonas fluorescens ATCC 39005 and Pseudomonas putida ATCC 39004. The aniline or aniline derivative thus produced was measured spectrophotometrically at 615 nm by a method similar to previously described methodologies using as an oxidizing agent a Cu II salt (or Fe III, chromate, dichromate or permanganate salt), a base in the form of a solution of ammonia and phenol or phenolic ether such as ortho-cresol.
These previous teachings were put to practice by Porton Products in a kit for the determination of acetaminophen in serum which comprises the sequential addition to serum of first an enzyme reagent followed by incubation for three minutes, a second addition of reagent A (1% ortho cresol solution) and a third addition of reagent B (ammoniacal copper solution). The color produced is read spectrophotometrically at 615 nm.
However, there are two problems associated with this methodology, (1) the three step addition of reagents makes the procedure cumbersome if done as a manual method and (2) the method cannot be conveniently automated in most instruments as it would require using three separate reagent channels to do one test plus a preincubation step prior to the addition of the two final reagents.
Accordingly, the present invention provides a simplified methodology for the determination of anilides by providing (a) a stabilized arylacylamidase enzyme preparation which can be conveniently integrated into a stable reagent to be used in any method for the determination of an anilide (b) a composition of reagents which can be made into one reagent so that the serum or matrix containing the drug can be added to it and measured spectrophotometrically in a one step reaction (c) by providing a format, as one reagent, which can be easily used with automated instruments by occupying only one channel as opposed to three channels and, (d) a stable composition of reagents that can be made into a solid-phase reagent test device.